Previous studies have consistently shown that caloric restriction (CR) decreases mitochondrial reactive oxygen species (ROS) (mitROS) generation and oxidative damage to mtDNA and mitochondrial proteins, and increases maximum longevity, although the mechanisms responsible for this are unknown. We recently found that protein restriction (PR) also produces these changes independent of energy restriction. Various facts link methionine to aging, and methionine restriction (MetR) without energy restriction increases, like CR, maximum longevity. We have thus hypothesized that MetR is responsible for the decrease in mitROS generation and oxidative stress in PR and CR. In this investigation we subjected male rats to exactly the same dietary protocol of MetR that is known to increase their longevity. We have found, for the first time, that MetR profoundly decreases mitROS production, decreases oxidative damage to mtDNA, lowers membrane unsaturation, and decreases all five markers of protein oxidation measured in rat heart and liver mitochondria. The concentration of complexes I and IV also decreases in MetR. The decrease in mitROS generation occurs in complexes I and III in liver and in complex I in heart mitochondria, and is due to an increase in efficiency of the respiratory chain in avoiding electron leak to oxygen. These changes are strikingly similar to those observed in CR and PR, suggesting that the decrease in methionine ingestion is responsible for the decrease in mitochondrial ROS production and oxidative stress, and possibly part of the decrease in aging rate, occurring during caloric restriction.—Sanz, A., Caro, P., Ayala, V., Portero-Otin, M., Pamplona, R., Barja, G. Methionine restriction decreases mitochondrial oxygen radical generation and leak as well as oxidative damage to mitochondrial DNA and proteins.